Human metabolic phenotypes link directly to specific dietary preferences in healthy individuals

Individual human health is determined by a complex interplay between genes, environment, diet, lifestyle, and symbiotic gut microbial activity. Here, we demonstrate a new "nutrimetabonomic" approach in which spectroscopically generated metabolic phenotypes are correlated with behavioral/psychological dietary preference, namely, "chocolate desiring" or "chocolate indifferent". Urinary and plasma metabolic phenotypes are characterized by differential metabolic biomarkers, measured using 1H NMR spectroscopy, including the postprandial lipoprotein profile and gut microbial co-metabolism. These data suggest that specific dietary preferences can influence basal metabolic state and gut microbiome activity that in turn may have long-term health consequences to the host. Nutrimetabonomics appears as a promising approach for the classification of dietary responses in populations and personalized nutritional management.     

Sustained nitric oxide production in macrophages requires the arginine transporter CAT2

The aberrant production of nitric oxide (NO) contributes to the pathogenesis of diseases as diverse as cancer and arthritis. Sustained NO production via the inducible enzyme, nitric-oxide synthase 2 (NOS2), requires extracellular arginine uptake. Three closely related cationic amino acid transporter genes (Cat1-3) encode the transporters that mediate most arginine uptake in mammalian cells. Because CAT2 is induced coordinately with NOS2 in numerous cell types, we investigated a possible role for CAT2-mediated arginine transport in regulating NO production. The complexity of arginine transport systems and their biochemically similar transport properties called for a genetic approach to determine the role of CAT2. CAT2-deficient mice were generated and found to be healthy and fertile in contrast to Cat1(-/-) animals. Analysis of cytokine-activated macrophages from Cat2(-/-) mice revealed a 92% reduction in NO production and a 95% reduction in l-Arg uptake. The reduction in NO production was not due to differences in NOS2 protein expression, NOS2 activity, or intracellular l-arginine content. In conclusion, our results show that sustained abundant NO synthesis by macrophages requires arginine transport via the CAT2 transporter.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Directly coupled high-performance liquid chromatography and nuclear magnetic resonance spectroscopic with chemometric studies on metabolic variation in Sprague--Dawley rats

We report here the first combined use of NMR-PR (pattern recognition) analysis and directly coupled HPLC--NMR analysis to identify metabolic subpopulations in normal laboratory animals and their discriminating endogenous urinary biomarkers. Urine samples obtained from control Sprague-Dawley rats (n = 68) were analyzed using (1)H NMR spectroscopy and principal components (PC) analysis to investigate physiological variability. Two distinct subpopulations of animals were classified based on metabolite excretion profiles. Analysis of the PC loadings established the spectral regions that were responsible for classification of the subpopulations and was used to direct the identification of biomarkers using a directly coupled HPLC--NMR analysis. One population had low urinary hippurate levels together with an increased concentration of 3-(3-hydroxyphenyl)propionic acid (3-HPPA)and 3-hydroxycinnamic acid (3-HCA). The other subpopulation excreted high levels of hippurate. Thus, we report the bimodal occurrence of hippuric acid and chlorogenic acid metabolites in a genetically homogeneous population of rats maintained under identical conditions, which may have significance in relation to the understanding of the consequences of biochemical variation in animals used for drug toxicity testing.     

Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain.

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[DeltadsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[DeltadsRBD] is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM). At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III[DeltadsRBD] is similar to that of RNase III at physiological salt concentrations and Mg(2+). In the presence of Mg(2+) RNase III[DeltadsRBD] is less efficient than the wild-type enzyme, due to a higher K(m). Similar to RNase III, RNase III[DeltadsRBD] is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[DeltadsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[DeltadsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[DeltadsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.     

EMG Reactivity and Oral Habits Among Facial Pain Patients in a Scheduled-Waiting Competitive Task

For individuals with temporomandibular disorder (TMD) it has been theorized that stressful events trigger oral habits (e.g., teeth grinding), thereby increasing masticatory muscle tension and subsequent pain. Recent research involving adjunctive behaviors found an increase in masseter surface EMG (sEMG) and oral habits when students with TMD symptomatology were placed on a fixed-time reinforcement schedule. The current study used a treatment-seeking community sample with TMD symptomatology in a competitive task designed to be a more naturalistic Fixed Time task. The experiment consisted of Adaptation, Free-Play, Scheduled-Play, and Recovery phases. During the Scheduled-Play phase participants played, and waited to play, an electronic poker game. Results indicated that masseter muscle tension in the Scheduled-Play phase was significantly higher (p<.001) than in any other phase. Moreover, during the Scheduled-Play phase masseter sEMG was higher (p#60;.001) when participants waited to play. Self-reported oral habits and overall affect were significantly higher (p's<.05) in the Free-Play and Scheduled-Play phases relative to Adaptation and Recovery. The observation that masseter sEMG was elevated during the Scheduled-Play phase relative to all other phases, and within the Scheduled-Play phase sEMG was highest while waiting, suggests that adjunctive oral habits may lead to TMD symptomatology.     

Caspase structure, proteolytic substrates, and function during apoptotic cell death

Caspases play an essential role during apoptotic cell death. These enzymes define a new class of cysteine proteases and comprise a multi-gene family with more than a dozen distinct mammalian family members. The discrete and highly limited subset of cellular polypeptides that are cleaved by these proteases is sufficient to account for the majority of cellular and morphological events that occur during cell death. In some cases, caspases also play a contributory role in escalating the propensity for apoptosis, and in doing so may exacerbate disease pathogenesis.     

Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes

We have produced mice that carry the human Ig heavy (IgH) and both kappa and lambda light chain transloci in a background in which the endogenous IgH and kappa loci have been inactivated. The B lymphocyte population in these translocus mice is restored to about one-third of normal levels, with preferential (3:1) expression of human lambda over human kappa. Human IgM is found in the serum at levels between 50 and 400 microg/ml and is elevated following immunization. This primary human Ab repertoire is sufficient to yield diverse Ag-specific responses as judged by analysis of mAbs. The use of DH and J segments is similar to that seen in human B cells, with an analogous pattern of N nucleotide insertion. Maturation of the response is accompanied by somatic hypermutation, which is particularly effective in the light chain transloci. These mice therefore allow the production of Ag-specific repertoires of both IgM,kappa and IgM,lambda Abs and should prove useful for the production of human mAbs for clinical use.     

Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168

Endospores of Bacillus spp. were purified from three Sonoran desert soil samples by Chelex extraction and NaBr density gradient centrifugation and their UV resistances compared with that of B. subtilis strain 168. Natural spore populations exhibited tight adherence to soil particles which was not readily overcome by the extraction and purification procedure. It was observed that spores purified from soil exhibited 2-3 fold higher resistance to UV (as measured by the 90% lethal dose, LD90) than did B. subtilis strain 168 grown on NSM, a standard laboratory sporulation medium, and purified by the same extraction procedure. Cultivation of spore-forming bacteria isolated from soil on NSM resulted in production of spores with essentially identical UV resistance as strain 168, suggesting that spore UV resistance is influenced by the environment in which spores are produced.